Background. Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation, which are limited by donor site morbidity and need for immunosuppression, respectively. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps and small diameter (i.e. small volume) reconstructions but may fail in larger defects due to a poorly-understood mechanism. ANAs provide endoneurial support but, due to removal of cellular content, depend on proliferation and migration of Schwann cells (SCs) from host tissue to support axonal regeneration. Populating larger-volume ANAs places a great proliferative demand on host SCs. This may stress or exceed the replicative limit of host SCs, resulting in senescence. In this study, we investigated the presence of senescent SCs (SenSCs) in long ANAs as a mechanism for failure of axonal regeneration.

Methods. Six Thy-1GFP rats underwent transection and repair with a 60 mm isograft (n=3) or ANA (n=3) to allow for in vivo visualization of axonal regeneration in long grafts as compared to shorter lengths (40 mm (n=2/group), 20 mm (n=2/group) after 10 weeks. Another twenty-six rats underwent sciatic nerve transection and repair with a 60 mm isograft (n=13) or ANA (n=13). Nerves were harvested after 10 weeks. Immunohistochemistry (n=9 per group) was used to determine the presence of SenSCs by staining for markers of SCs (S100) and cellular senescence (?-galactosidase, p16INK4A). Quantitative PCR analysis (n=4 per group) was also used to quantify levels of SCs and expression of senescence markers (p16INK4A , p53, and IL-6) within grafts. Lastly, the SCs from a representative nerve from each group were examined under electron microscopy (EM) for senescence-associated changes in chromatin.

Results. In vivo imaging of nerve grafts in Thy1-GFP rats demonstrated a strong inverse relationship between the length of ANA graft and axonal regeneration. Immunohistochemical staining of grafts for markers of senescence revealed a significantly increased concentration of SenSCs in the ANA, in an area corresponding to just distal to the stalled axonal front in the 60 mm graft. Furthermore, SCs in this region exhibited reorganization of chromatin that is associated with senescence.

Conclusions. There is significant evidence to suggest that cellular senescence contributes to the inability of host SCs to support axonal regeneration across long ANAs. Prevention of SC senescence may enhance regeneration through large volume ANAs.